Male reproductive disorders account for 40% of infertility cases worldwide in humans. Although causes of infertility are multi-factorial, exposure to estrogens (E) during development has been linked with increasing incidence of reproductive disorders. Mechanisms underlying E-induced disorders remain unclear. The goal of this study is to determine molecular and cellular mechanisms whereby neonatal E exposure results in infertility and mal-developed penis characterized by abnormal accumulation of fat cells and loss of smooth muscle and blood vessels in the penis body. The central hypothesis is that E exposure, via estrogen receptor (ER) pathway or androgen receptor (AR) pathway or both, alters ER1 expression in penile stromal cells (higher E, higher ER1; lower androgen, higher ER1), which are then re-programmed so that key genes for differentiation of fat cells (PPAR3, C/EBP1) are undesirably up-regulated and those for smooth muscle cell (1 actin) and endothelial cell (CD-31) are down-regulated. Specific aim 1 will test the hypotheses that I) E exposure up-regulates ER1 expression and down-regulates neonatal testosterone surge in a dose- dependent manner, II) ER1 up-regulation is time-dependent and occurs during a critical developmental period; and III) ER1 up-regulation is mitigated by ER antagonist ICI 182,780 (ER pathway), as well as by AR agonist DHT (AR pathway). Specific aim 2 will test the hypotheses I) that E exposure up-regulates PPAR3 and C/EBP1 expression and down-regulates 1 actin and CD-31 expression (cause and effect relationships); and II) that ICI and DHT mitigate these alterations. Specific aim 3 will test the hypothesis that exposure to anti-androgens (GnRH-antagonist and flutamide) up-regulates ER1 expression because of lower androgenic action, but may not alter PPAR3, C/EBP1, 1 actin, and CD-31 expression because of lack of exogenous E exposure. Specific aim 4 will test the hypothesis that intervention with ER antagonist and AR agonist prevents E-induced loss of fertility. Real-time PCR will be used to quantify mRNAs and immunohistochemistry will be used to localize and quantify proteins. Collectively, results will unravel mechanisms whereby altered signaling in ER pathway or AR pathway or both results in mal-development of the penis and penile dysfunction. Outcomes of this work will have a significant impact on human and wildlife health because they will lead to better strategies to prevent E-induced reproductive disorders. PUBLIC HEALTH RELEVANCE: Exposure to environmental estrogens (endocrine disruptors) has been linked with increasing frequency of reproductive disorders, underscoring their global risks in human and wildlife health. The objective of this application is to determine molecular and cellular mechanisms whereby neonatal estrogen exposure results in permanent loss of fertility and mal-developed penis characterized by accumulation of fat cells and loss of smooth muscle cells and blood vessels in the body of the penis.